Preservation



y 9 c. H. BARR 2,353,986

' PRESERVATION Original Filed Nov. 7, 1938 2 Shee'ts-Sheet l I I I l I vQ 26 46 a I: (72 INVENTOR.

. CouRTifM D h; BAR i July 1944- v c. H. BARR 2,353,986

wnnsmwulbn Y I f 7 Original Filed Nov. 7, 1958 2 Sheets-Shet 2 a O v QINVENTOR. (CORTLAND 54m; BY

/ ff iww Patented July 18, 1944 PRESERVATION Courtland H. Barr,Glenoiden, Pa assignor to Sharp lb Dohme Incorporated, Philadelphia,Pa., a corporation of Maryland Original application November 7, 1938,Serial No.

Divided and this application August a, 1943, Serial No. 497,251 (01.34-5) Claims.

This invention relates to improvements in processes-and apparatus forthe preservation of biologically active and other substances, includingsuch substances as sera, protein solutions, bacterial cultures,pharmaceutical and glandular substances, viruses and other labilebiological substances. It relates more particularly to improvements inprocesses and apparatus for sealing such substances in final containersunder a high vacuum after desiccating such substances by The presentinvention provides improved processes and apparatus which permit theproduction of desiccated biologically active and other substances incontainers with perforable stoppers, sealed with glass seals, with theperforable stoppers, such as-rubber stoppers, sealed within, the glass,the perforable stoppers'being positioned 7 while the .originual vacuumis maintained in the freezing them and removing'water under a highvacuum. v

The improved processes and apparatus of the present invention permit thedesiccation of such biologically active substances, with subsequentsealing in a glass or other container with a perforable stopper, understerile conditions and with rigid control, with the simultaneousproductionof a large number of ackages containing the material suitablefor distribution and use.

Desiccated biologically active substances, produced in desiccated formby processes involving freezing of the original liquid material followedby removal of water by the application of a high vacuum without meltingof the frozen material, are very porous and hygroscopic. It is desirableto package and distribute the material under a vacuum, both to preventany deterioration, contamination or absorption of water, all of whichmight be caused by contact with air, and to facilitate the propersolution of the material in water or other aqueous fluid whenrestoration to a liquid state is desired. If such material is maintainedunder a vacuum, and water is introduced into the container before thevacuum is broken, the vacuum tends to pull the water into the pores andinterstices of the material, insuring intimate contact and wetting ofthe material with the water, allowing rapid solution of the material.Maintenance of the vacuum also prevents impregnation of the materialwith gas which, if present, would tend to prevent the ready access ofwater to the interior portions of the material and to slow up itsrestoration to a liquid state.

Further, it is very desirable to seal the material within the containerwhile continuing the same vacuum used in processing the material,without the introduction of air or other gas from the beginning of thedesiccation to the final vacuum sealing, because of the greaterassurance that if the material is sterile initially, it will be sterilewhen finally sealed in the container. Nothing is introduced into thecontainer after the processing begins.

containers,so that the material within the containers is sealed underthe original vacuum, with assurance that the material is free fromcontamination, air, moisture, etc. The apparatus is such that a largenumber of such final containers, for example, 100 or more, may bereadily processed at one time, with uniform dehydration of the materialwithin the various containers, and with proper sealing of all of thecontainers after the desired degree of desiccation is attained.

In carrying out the invention, the biological or other substance to bedesiccated is introduced in a liquid state into a suitable vial or otherfinal container, preferably made of ordinary soft glass, and cylindricalin shape, provided with a relatively long tubular neck, the. inside ofwhich at the point adjacent the top of the vial or container isadvantageously tapered to fit a rubber stopper. In general, the quantityof material introduced into the container should not exceed about of thecapacity of the container, to permit an ample surface for thesublimation or vaporization of the ice. The size of the vials.

or containers used may be widely varied, their capacity being determinedby the amount of material which they are intended to contain andadvantageously being such that each contains one or more unit portionsof the material.

centimeter to 50 or 100 or more cubic centimeters. After theintroduction of the liquid substance into the container, the material isfrozenby exposing the container to a refrigerant maintained at a verylow temperature. The freezing is advantageously carried out while thecontainer is I being rotated about its lonigtudinal axis while .in amore or less horizontal position, to insure a maximum exposed surfacefor sublimation; Of

Thus the capacity may vary from a fraction of a cubic placed, wh le openand under sterile conditions, in suitable apparatus, such as that ofthis invention, which has been previously sterilized, a high vacuum isapplied simultaneously to all of the containers and the desiccation iscarried out.

After the desiccation has proceeded to the desired extent, a rubberstopper is introduced in the neck of each of the containers, 'and isforced down to form a tight joint in the tapered or constricted portionof the neck. This introduction of the stoppers is carried out withoutbreaking the vacuum, by externally actuated means, so that the materialis sealed in each container under a high vacuum with a perforableclosure and without contamination, impairment of sterility, etc.

As a relatively large number of the containers are processedsimultaneously in a vacuum chamber, and under substantially identicalconditions, with the exterior and interior surfaces of the containers,as well as the stoppers, subjected to the dehydration, there isassurance that the product obtained conforms quite closely to thestandards which may be set as to the degree of desiccation, vacuumwithin the containers, etc.

The process of the present invention provides for the dehydration of thebiologically active or other substance in final containers in a suitablevacuum chamber, with simultaneous removal of water from the material ina large number of such containers and simultaneous sealing of suchcontainers with perforable stoppers without breaking the vacuum andunder the original vacum used in the processing. This process may becarried out by placing a large number of containers in a suitablechamber under sterile conditions and producing a high vacuum within thechamber to effect a rapid evaporation or sublimation of the frozen waterin the product until the desired degree of desiccation is attained. Asthe containers open directly into the evacuated chamber, there is nosubstantial resistance to now of vapors except such as is offered by thecontainer necks, which, of course, are of sufllcient diameter to permitfree flow. After the desiccation is completed, and without breaking ordestroying the vacuum within the chamber, a perforable stopper isintroduced into the neck of each container, and is forced into tightengagement with the neck of the container, which is advantageouslytapered as explained.

It is advantageous to supply heat to the material undergoingdesiccation, as by jacketing the vacuum chamber at least over the lowerportions thereof, with a controllable heating bath, so that thedesiccation is not unduly prolonged because of the refrigerating effectof the vaporization or sublimation. After the stoppers have beenproperly seated, air is introduced into the chamber, advantageouslythrough an air filter to sterilize it, the containers are removed fromthe chamber and the necks are flame sealed above the stoppers.

The apparatus of the invention includes a chamber provided with aremoval wall section adapted to be closed vacuum-tight and with anoutlet leading to one or more vacuum condensing systems, as will besubsequently explained. There is also provided a rack or othersupporting means, advantageously positioned at the bottom of thechamber, adapted to be placed within the chamber to support a largenumber of containers in more or less fixed positions, so that thecontainers rest on the bottom of the chamber with their necks extendingupwardly. A corresponding number of plungers, suitably supported, areprovided above the rack, so located that when in their normal position,each plunger has beneath it a. container. The size of the plungers issuchthat they may readily pass into the tubular necks of the containers.Guides are provided for each of the plungers so that when the plungersare forced downwardly toward the containers, each plunger will enter theneck of the corresponding container. Below the plungers are provided acorresponding number of sleeves or other supports, which may surroundthe lower portions of the plungers and serve as their guides, each ofwhich is adapted to support a perforable stopper. The plungers areadvantageously supported at their top by common supporting means, suchas a disc or plate, such that when the disc or plate is forceddownwardly, each of the plungers is also forced downwardly to disengagethe stoppers from the sleeves or other supports, and to force thestoppers into place in the tapered portions of the necks of thecontainers. The rack for supporting the containers, the plungers, andthe sleeves or other supporting means for the stoppers, together withguides which are advantageously provided to hold the upper portions ofthe containers in position, are advantageously fastened to or supportedon a single central axis, so that the entire assembly may be removedfrom the chamber and handled as a unit, for sterilizing or otherpurposes.

It is also advantageous to provide a heating bath around the chamber, toprovide a regulated flow of heat through the chamber walls to the frozenmaterial within the containers, to supply the latent heat ofvaporization or sublimation of the ice and thus to increase the rapidityand degree of the desiccation. The rate at which heat is supplied shouldnot be so great as to cause melting or softening of the frozen material,or so low as to impede or slow up the process of desiccation, but shouldbe such as to give a reasonably rapid removal of water, without meltingor softening.

The invention will be further described in connection with theaccompanying drawings, which illustrate, in a somewhat diagrammaticfashion, apparatus which embodies the invention and which isadvantageous for use in the desiccation of biological products.

In the drawings,

Fig. 1 is a view in elevation of the apparatus, showing in verticalsection the vacuum chamber, the plunger actuating means and the heatingbath;

Fig. 2 is a vertical sectional view of the removable assembly which fitswithin the chamber, showing the containers and stoppers in place; and

Fig. 3 is a sectional view in elevation of a portion of the assembly ofFig. 1, showing in somewhat greater detail a method of supporting thecontainers and the stoppers.

In Fig. 1, there is provided a chamber 10, with a bottom [2, side wallsH and a removable cover plate l6, fastened to the walls I by flanges IIand bolts 20, with the rubber gasket 22 to insure a vacuum-tight seal.The bolts 20 may be omitted, if desired, particularly where the coverplate forms the top walls, or a portion of it, as the weight of thecover plate is usually sufficient to permit evacuation without muchinflux of air, and, when the chamber is evacuated, the atmosphericpressure on the cover plate is suflicient to hold it in vacuum-tightengagement with the flange of the vacuum chamber.

Around the chamber I6 is provided a heating bath 24, containing a heatconductive medium 26 and heating coils 26. The temperature of theheating bath should be regulated by a suitable .thermostat, or othercontrol means (not shown).

A cover 66 for the heating bath is provided. Other means of supplyingheat in controlled amounts may, of course, be used. For example, insteadof providing heating coils 26 in the heating bath, the heating mediummay be withdrawn and heated by outside means, or a continuous flow ofproperly heated medium through the heating bath may be provided, or theheating bath may be connected to a vessel containing properly heatedheating medium, below or adjacent the heating bath, with circulation toor from the heating bath depending on convection v currents, etc.

The chamber I6, and the heating bath 24, are supported on the base plate62. The chamber I6 is supported somewhat above the bottom of the heatingbath by the legs 34, which rest on the bottom of the heating bath, sothat the chamber I6 in effect is directly supported by the base plate62.

The chamber I6 is provided with an outlet 36, which in turn is providedwith two branches, l6 and 46, each branch and the outlet 66 beingprovided with a vacuum tight valve, 4i, 42 and 46. Outlet 36 isconnected to an air filter 44, so that sterile air may be introducedinto the chamber after tightly closing the containers with stoppers.Outlet 36 leads to a condenser and vacuum system, and outlet 46 toanauxiliary condenser and vacuum system, as will be hereinafterexplained.

Positioned within the vacuum chamber I6 is the removable containersupporting and plunger mechanism, shown in section in Fig. 2. Thisincludes a bottom supporting plate 46 to which is fastened a centralpillar 46. The plate 46 is provided with a series of depressions 56,adapted to receive the containers. Above the bottom plate 46 may beprovided a second plate 52, fastened to the sleeve 54 and provided withholes corresponding to the depressions 56 in the plate 46, to assist inholding the containers in proper alignment. Above plate 52 may beprovided a second plate 56, readily detachable, having sleeves 56, whichserve to adapt the size of the holes in the plate 52 and the depressions56 in the plate 46 to the size of the containers used. If desired, theplate 56 may be provided with cups instead of the sleeves 56, to adaptthe apparatus for use with small sized containers. The sleeve 54 isrigidly connected to the plate 46, and is around a second, shortersleeve 66, which is removable.

At the periphery of the plate 46 are provided posts 62, advantageouslyof adjustable height, as illustrated. Advantageously three or more ofthese posts are provided, because they serve to fix the position of thecontainer aligning plates 52, 56 and 64, which fit over these posts. Theposts are made adjustable by providing a short inner fixed post 66 andremovable upper sections 66 held in place by the removable sleeve I6 andfitting over the lug I2.

The posts 62 serve not only to position correctly the plates 52 and 56,but to support and position the plate 64, which is provided withdownwardly extending cones I4, which flare downwardly from holescentered vertically above the holes 56 in the plate 46. Plate 64 servesto hold the necks of the containers in the proper position, and isprovided with the conical elements 14 to simplify passing the plate 64over the container. necks after the containers have been placed inposition on the plate 46. Posts 62 are made of adjustable height so thepositionsof plate 64 can be varied to accommodate containers ofdifl'erent sizes. In general, all of the containers processed in oneoperation will be of the same height, and the position of the plate 64need be varied only when the size of the container being used ischanged.

Above the plate 64 is positioned another plate 16, supported by thesleeve I6, which in turn is supported by the sleeve 46, both sleeve 66and sleeve I6 fitting within sleeve 64. Sleeve I6 is provided with a key66 which fits a corresponding key-way in the sleeve 54 to insure properposi-.

tioning of the plate I6. The plate I6 is provided with holes 6|, beneathwhich are the sleeves 62, advantageously provided with the flaringportions 64. These holes and sleeves are in alignment with the holes inthe plates 52, 56 and 64 and the depressions 56 in the plate 46. Thelength of the sleeve 66 and the sleeve I6 are such that the sleeves 62extend downwardly to a point just above the top of the necks of thecontainers,

advantageously with the flaring portions 64 extending a little below thetops of the necks of the containers. Ample space above and around thetops of the container necks to permit free flow of vapor from thecontainers should be provided. Holes or slots 63, in the sleeves 62, areadvantageously provided to decrease the resistance to the flow of vapor.By changing the length of the sleeve 66, the position of the plate I6may be ad- 'Justed to accommodate containers of different .of the rods94 is such that in normal position,

they extend down to about the top of the stoppers 96. The rods 94, areof course, of uniform length. Attached to the holding flange 92 is asleeve 99, which serves to support the spring 66, this sleeve extendingdownwardly but a portion of the way past the top of the sleeve 16.Sleeves 62 should have a smaller inner diameter at the top than at thebottom and the lower ends of the rods 94' should be expanded, orprovided with buttons as illustrated, to prevent the plungers frombecoming disengaged from the guiding sleeves, and to prevent the spring68 from forcing the plate 96 too high.

The entire assembly, as described, may be lifted by the handle 3|, whichmay be detachably fastened to the central post 46, and the assembly maythus be moved as a unit, and removed from, or placed within, thecontainer l6 as a unit;

At the top of the container I6 is provided a rod I66, which is movablevertically. This rod is provided at the bottom with a detachable blockI62, which when the apparatus is assembled, bears against the holdingflange 92. At the top of the movable rod I66 is provided a fixed flangeI64, rigidly fastened to therod I66. Rigidly fastened to the cover I6 isthe flange I06, through which the rod I passes. To provide avacuum-tight seal, rubber tube I00, supported by the spiral spring IIOextends from flange I04 to flange I 08, and tightly embraces both. Toprovide this seal, there may be provided grooves I05 in flanges I04 andI00 and cooperating clamping rings or wires I01 around the rubber tubeI08.

Extending from the base 32 are the tie rods I I2, which in turn areremovably fastened to the yoke M4 by the nuts H5, in effect forming amechanical press. Passing through the yoke II4 is the screw rod IIS, towhich is fastened the handle H8; The screw rod bears against the upperportion of the vertically moving rod I00 by a suitable bearing surfaceI20.

By turning the handle II8, the rod I00 may be forced downwardly, in turnforcing downwardly the plate 90, displacing the stoppers from.

the sleeves 82 into the necks 0f the containers. Further downwardmovement of the rod I00 forces the plungers 94 into the necks of thecontainers, so that they force the stoppers securely into position inthe tapered portions of the necks of the containers.

The lengths of the adjustable posts 52, sleeve 60, the block I02, andthe distance the rod I00 After the high vacuum is established, and thedesiccation is under way, with water being absorbed in the condenser orother water absorbing means connected to outlet 40, the valve 42 isopened, and the valve 43 is closed. Outlet 84 leads to a main watercondenser or absorber and vacuum pumnto which may be connected a numberof other desiccating units. The flow of gas and vapor is always out fromthe chamber I0 into the condensing and vacuum systems, so that after thechamber is initially sealed no air or gas can flow into it prior to thesealing. of the containers to carry in foreign matter which mightcontaminate the material being treated.

The vaporization or sublimation of the ice from the frozen materialexerts a considerable cooling effect, and if heat is not supplied Sllfile y fast.

. the heat. of vaporization or sublimation, with is moved must beadjusted to properly accommodate the containers used, so that theplunger rods 94 force the stoppers the proper distance into thecontainer necks. It is to be understood that in any given operation, thecontainers and stoppers will be uniform, but the apparatus may bereadily adjusted for use with different size containers by making properadjustments in the various parts, all of which are detachable andreplaceable.

In operation, the entire assembly supported by the plate 46 is removedfrom the container I0, and is sterilized as a unit, or with the upperportions removed and separately sterilized. Containers with frozenmaterial in them, produced as heretofore explained, are placed in properposition on the plate 40, the aligning plate 64 is placed in position,after which the plate I6 with the stoppers in place in the sleeves 82(these are advantageously placed in the sleeves prior to thesterilizing) as a unit with the plate 90 and the rods 94 dependingtherefrom through the sleeves 82 are placed in position. Theseoperations must, of course, be carried out with sterile technique; inaccordance with usual practice. The entire assembly is then lifted, asby the handle 3|, and placed in the container I0. The handle is thenremoved, the desired detachable block I02 is set into the flange 92 (seeFig. l) and the cover I5 is fastened in place by the bolts 20. Valves H,42 and 43 are closed.

Valve 43, on outlet 40, which leads to a suitable condenser or waterabsorbing apparatus and to a high vacuum pump, which need not have avery large capacity, is opened, the air is removed from the container I0and a high vacuum established therein, causing a more or less rapidsublimation or vaporization of the frozen material in the containers.This operation of placing the containers in position, and assembling theapparatus, must be carried out with sufficient rapidity to make surethat the desiccation is under way before any of the frozen materialmelts or softens, so that all removal of water is from solidly frozenmaterial. After the vaporization or sublimation has begun, the heatingbath may be placed in operation, to supply heat to the con. tainers tofacilitate the dehydration.

maintenance of the frozen material at a temperature such that thevaporization is reasonably rapid, but sumciently low so that it does notmelt or soften. After most of the water is removed from the frozenmaterial, so that even at ordinary temperatures it has a dry appearance,the temperature of the material is allowed to rise to facilitate theremoval of the last traces of water. By so operating, a productcontaining one-half of one percent or less of water is readily obtained,such products having about 99.95% or somewhat more of the original waterremoved. In general, heat should be supplied as fast as possible withoutcausing melting or softening of the frozen material.

By using two vacuum connections, a connection to a main condensing andvacuum system, and a connection to an auxiliary condensing and vacuumsystem, it is possible to use one large condensing and vacuum system fora number of such units of apparatus such as have been described, so thatone or more units may be added to, or removed from, the system withoutdisturbing the conditions in the main condensing and vacuum system, asapparatus is connected to the main system only when the conditions in itare substantially those of the main system.

Insofar as the process of freezing and removing the water is concerned,the process which is advantageously used is one described in the ReichelPatent Re. 20,969 with freezing of the material by indirect exposure toa refrigerant maintained at -'70 C. or lower, and with condensation ofthe water vapor removed under the influence of a high vacuum by exposureto a refrigerant maintained at a similarly low temperature, although itwill be appreciated that other means of collecting the water vapor, etc.may be used. When such a process is used, each outlet is connected to acold condenser maintained at a sufficiently low temperature, forexample, -50 C. or lower, which in turn is connected to a vacuum pump.The water vapor is condensed in the cold condenser and the vacuum pumpremoves fixed gases and maintains the high vacuum necessary forsublimation or vaporization. Two such condensing and vacuum systems areadvantageously provided, one of large capacity and one which may be ofsmaller capacity, to facilitate the operation of a number of suchdesiccating units as have been described. The unit is first connected tothe smaller (auxiliary) svstem to evacuate it and to initiate thedesiccation, and subsequently is connected to the main system, by whichthe majorportion of the water is removed. It should be understood thatother water absorbing means than cold condensers, such as chemicaldesiccants. etc., may be used if desired. The condensing. or waterabsorbing system forms no part of the present invention.

After the desiccation has proceeded to the desired extent, and thestoppers have been forced into the container necks, the valve 42 isclosed, and sterile air is admitted to the chamber through the filter44, line 38 and valve 4|. The cover is then removed, and the containerstaken out and flame-sealed above the stoppers, so that a container, witha perforable stopper in the neck, and a flame seal above the stopper, isobtained. Such containers, and methods of sealing the necks, aredescribed in my prior application, Serial No. 90,033, filed July 10,1936. It will be noted that the containers are sealed by the perforablestoppers under the vacuum used in processing the material, so that therecan be no contamination of the material during the processing, or afterthe processing and before the sealing.

It will be thus understood that the present invention provides improvedprocesses and apparatus by which a large number of packages containing adesiccated biologically active or other substance sealed'under a highvacuum with a perforable stopper, with a glass seal over the perforablestopper, may be processed simultaneously, with assurance of sterilityand with a minimum of labor.

This is a division of my application Serial No. 239,417, filed November7, 1938.

I claim:

1. The method of drying a product containing water comprising freezingsaid product, subjecting said i'rozen product to a vacuum effective tosublime the ice without permitting it to pass through an intermediateliquid phase and for a period sumcient to remove most 01' the waterwhile maintaining the temperature of said frozen product below itsfreezing temperature, and thereafter raising the temperature of saidfrozen product while the residual water is being removed.

2. The method of drying aproduct containing water comprising freezingsaid product, subjecting said frozen product to a vacuum effective tosublime the ice without permitting it to pass through an intermediateliquid phase and for a period suiflcient to remove most of the water,sup-.

plying heat to said product in an amount to equal the cooling eil'ect ofsuch sublimation, and thereafter raising the temperature of said productwhile the residual water is being removed.

3. The method of drying a product containing water comprising freezingsaid product, subjecting said frozen product to a vacuum effective tosublime the ice without permitting it to pass through an intermediateliquid phase and for a period suflicient to remove most of the water,supplying heat to said product in an amount to equal the cooling effectof suchsublimation while maintaining the temperature of said frozenproduct below its freezing temperature, and thereafter raising thetemperature of said product while the residual water is being removed.

4. The process as in claim 1, in which the vacuum is appliedsimultaneously to a number of containers and materials placed in achamber by evacuation of said chamber.

5. The process as in claim 2, in which the vacuum is appliedsimultaneously to a number of containers of material placed in a chamberby evacuation of said chamber.

COUR'ILAND H. BARR.

